The focus of our research is the determinants of two transcriptional properties of steroid receptors: the dose-response curve of agonists and the partial agonist activity of antisteroids. The dose-response curve defines the EC50, or steroid concentration at which half-maximal response is seen, and is a crucial but poorly understood component of steroid hormone endocrinology. The partial agonist activity of antisteroids is an important consideration for limiting unwanted side effects during endocrine therapies. While these two properties were long considered to be invariant, mounting evidence indicated that they could be varied to provide a mechanism for differential control of gene activation. The first modulator to be described for these two transactivation properties of any steroid receptor was the glucocorticoid modulatory element (GME), which we identified as an upstream DNA sequence in the glucocorticoid-inducible tyrosine aminotransferase gene. Subsequently, we found that two new proteins bound to the GME. In an effort to understand the mechanism of action of the GME, we have characterized one of these two new proteins (GMEB-1) and a biologically active protein that binds to GMEB-1, i.e., Ubc9. At the same time, basic studies on steroid binding to glucocorticoid receptors (GRs) have revealed a completely different pathway for modulating GR transcriptional properties. GMEB-1 was initially described as being a component of a 550 kDa heteromeric DNA-binding complex that is required for GME activity. In order to understand the role of GMEB-1 at a molecular level, we determined which GMEB-1 sequences are associated with the various activities associated with the modulation of GR transactivation properties. These activities include, homooligomerization, heterooligomerization, DNA binding, binding to GR and the transcriptional cofactor CBP, and GR modulation itself. Complex activities, such as DNA binding and GR modulation, are found to require the physical combination of those domains that would be predicted from the involved biochemical processes. We previously documented that GMEB-1 possesses both GR modulatory and intrinsic transactivation activity. However, the domains for these two activities of GMEB-1 are found not to overlap. This separation of activities provides a structural basis for our prior biological observations that the modulation of the dose-response curve, and partial agonist activity, of GR complexes is independent of the total levels of gene activation by the same GR complexes. A yeast two-hybrid assay was employed to identify proteins that bind to GMEB-1 and could be involved in the modulatory actions of the GME. One interacting protein is Ubc9, which is the mammalian homolog of a yeast E2 ubiquitin-conjugating enzyme and often transfers the molecule SUMO-1 to target proteins. Unexpectedly, Ubc9 also binds to GMEB-2, the homooligomerizing partner of GMEB-1, and to GRs. Ubc9 displays no intrinsic transactivation activity but modifies both the absolute amount of induced gene product and the fold induction by GRs. With high concentrations of GRs, added Ubc9 reduces the EC50 of agonists and increases the partial agonist activity of antagonists in a manner that is independent of the ability of Ubc9 to transfer SUMO-1 to proteins. This new activity of Ubc9 requires only the ligand binding domain of GR, and part of the hinge region. Interestingly, Ubc9 modulation of full length GR transcriptional properties can be seen in the absence of the GME. This is consistent with the GME acting by increasing the local concentration of Ubc9, which then activates a previously unobserved target in the transcriptional machinery. With high concentrations of GRs, Ubc9 binding to GRs appears to be sufficient to permit Ubc9 to act independently of the GME. Concomitant studies on the details of steroid binding to GRs uncovered an entirely different means of modulating GR transactivation properties. The association of heat shock protein 90 (hsp90) with GR has long been known to be required for the steroid binding activity of GR. We had earlier reported that seven amino acids (547-553) overlapping the amino terminal end of the rat GR ligand binding domain (LBD) are necessary for hsp90 binding, and consequently for steroid binding. We have now conducted saturation mutagenesis of this sequence, which appears to be part of the surface where the ligand binding cleft merges with the surface of the LBD. No single point mutation causes significant changes in any of a variety of biochemical and biological properties in addition to hsp90 binding. A triple mutation (P548A/T549A/V551A) increases the EC50 by >100-fold without affecting the level of maximal induction or coactivator response. Interestingly, this triple mutant displays reduced binding of steroid and hsp90 in whole cells but possesses wild type affinity for steroid and normal hsp90 binding capacity under cell-free conditions. This phenotype of a dramatic shift in the dose-response for transactivation would be expected from an increase in the rate of disassembly of the triple mutant GR?hsp90 heterocomplex in the cell. Mutation of the entire 7-amino acid region to CAAAAAC maintains the presence of a critical a-helical structure and heterocomplex formation with hsp90 but eliminates steroid binding and transcriptional activation, thus disconnecting hsp90 binding from opening of the ligand binding cleft and steroid binding. As a result of the above studies, we have gained detailed information about two disparate processes for modulating GR transcriptional properties. The first involves Ubc9, which binds to two proteins (GMEB-1 and -2) that are required for the activity of the first modulatory factor (the GME) and which appears to interact with a component of the transcription complex. The second process affects a much earlier step, i.e., hsp90 binding to GRs, which regulates the steroid binding activity of GRs. The existence of two unrelated modulatory pathways, coupled with our previous demonstration that the transactivation properties of progesterone receptors can also be modulated, further supports our hypothesis that the modulation of the EC50 and the partial agonist activity of steroid-bound receptors is of general and widespread importance for the differential control of gene expression during development, differentiation, and homeostasis. These combined findings contribute to our long term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.